Identification and evaluation of Escherichia coli strain ATCC 8739 as a surrogate for thermal inactivation of enterohemorrhagic Escherichia coli in fruit nectars: Impact of applied techniques on the decimal reduction time.

The objective of this study was to identify a suitable surrogate for E. coli O157:H7 strain 19685/91 and O113:H21 strain TS18/08, by assessing their thermal resistance at temperatures of 60 °C, 65 °C, and 72 °C in strawberry nectar. The influence of the matrix and the research methodology on the decimal reduction time (D-value) was investigated. Thermal kinetics and safety assessment demonstrated that E. coli ATCC 8739 is a suitable surrogate. The study demonstrated that the presence of fruit particles in the nectar increased thermal resistance of the tested strains. Variations in D-values were observed depending on the research method employed, with D-values in glass capillaries were up to 6.6 times lower compared to larger sample volumes. Encapsulation of E. coli ATCC 8739 exhibited high efficiency of 90.25 ± 0.26% and maintained stable viable counts after 26 days of storage in strawberry nectar at 4 °C. There were no significant differences in thermal resistance between surrogates directly inoculated into strawberry nectar and those encapsulated in alginate beads. Additionally, the encapsulated strains did not migrate outside the beads. Therefore, encapsulated E. coli ATCC 8739 in alginate beads can be effectively utilized in industrial settings to validate thermal treatments as a reliable and safe method.

RNA-binding protein Hfq downregulates locus of enterocyte effacement-encoded regulators independent of small regulatory RNA in enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) causes severe human diseases worldwide. The type 3 secretion system and effector proteins are essential for EHEC infection, and are encoded by the locus of enterocyte effacement (LEE). RNA-binding protein Hfq is essential for small regulatory RNA (sRNA)-mediated regulation at a posttranscriptional level and full virulence of many pathogenic bacteria. Although two early studies indicated that Hfq represses LEE expression by posttranscriptionally controlling the expression of genes grlRA and/or ler, both of which encode LEE regulators mediating a positive regulatory loop, the detailed molecular mechanism and biological significance remain unclear. Herein, we show that LEE overexpression was caused by defective RNA-binding activity of the Hfq distal face, which posttranscriptionally represses grlA and ler expression. In vitro analyses revealed that the Hfq distal face directly binds near the translational initiation site of grlA and ler mRNAs, and inhibits their translation. Taken together, we conclude that Hfq inhibits grlA and ler translation by binding their mRNAs through the distal face in an sRNA-independent manner. Additionally, we show that Hfq-mediated repression of LEE is critical for normal EHEC growth because all suppressor mutations that restored the growth defect in the hfq mutant abolished hfq deletion-induced overexpression of LEE.

Variability in growth responses of non-O157 EHEC isolates in leafy vegetables, sprouted seeds and soil extracts occurs at the isolate level.

Foods of plant origin are recognised as a major source of foodborne pathogens, in particular for Shigatoxigenic Escherichia coli (STEC). Most work for STEC and plant-based fresh produce has focused on the most prevalent outbreak serogroup, O157. However, non-O157 STEC is an emerging hazard, and as such it is important to characterise aspects within this group that reflect their ability to colonise alternative hosts and habitats relevant to horticultural production. Growth kinetics were quantified for a diverse set of clinical enterohaemorrhagic E. coli isolates in extracts made from different tissues of spinach, lettuce or sprouted seeds, or from soil, to represent association with ready-to-eat fresh produce production. For leafy vegetables, spinach apoplast supported the fastest rates of growth and lettuce root extracts generated the slowest growth rates. Growth rates were similar for the majority of isolates in fenugreek or alfalfa sprouted seed extracts. Monosaccharides were the major driver of bacterial growth. No correlations were found for growth rates between different serotypes or for Shigatoxin gene carriage. Thus, growth rates varied in a plant-dependent and isolate-dependent manner, for all plant or soil extracts tested, indicative of isolate-specific differences in metabolic flexibility. These findings are relevant for risk assessment of non-O157 STEC.

Effect of Vacuum Steam Treatment of Hard Red Spring Wheat on Flour Quality and Reduction of Escherichia coli O121 and Salmonella Enteritidis PT 30.

ABSTRACT: Recent outbreaks traced to contaminated flour have created a need in the milling industry for a process that reduces pathogens in wheat while maintaining its functional properties. Vacuum steam treatment is a promising technology for treatment of low-moisture foods. Traditional thermal treatment methods can compromise wheat functionality due to high temperatures; thus, maintaining the functional quality of the wheat protein was critical for this research. The objective of this study was to evaluate the effect of vacuum steam treatment of hard red spring (HRS) wheat kernels on final flour quality and the overall efficacy of vacuum stream treatment for reducing pathogens on HRS wheat kernels. HRS wheat samples were treated with steam under vacuum at 65, 70, 75, and 85°C for 4 and 8 min. Significant changes in dough and baked product functionality were observed for treatments at ≥70°C. Treatment time had no significant effect on the qualities evaluated. After determining that vacuum steam treatment at 65°C best preserved product quality, HRS wheat was inoculated with Escherichia coli O121 and Salmonella Enteritidis PT 30 and processed at 65°C for 0, 2, 4, 6, or 8 min. The treatments achieved a maximum average reduction of 3.57 ± 0.33 log CFU/g for E. coli O121 and 3.21 ± 0.27 log CFU/g for Salmonella. Vacuum steam treatment could be an effective pathogen inactivation method for the flour milling industry.

The protective effects of enriched citrulline fermented milk with Lactobacillus helveticus on the intestinal epithelium integrity against Escherichia coli infection.

This study examined the protective effects of citrulline enriched-fermented milk with live Lactobacillus helveticus ASCC 511 (LH511) on intestinal epithelial barrier function and inflammatory response in IPEC-J2 cells caused by pathogenic Escherichia coli. Five percent (v/v) of fermented milk with live LH511 and 4 mM citrulline (5%LHFM_Cit-4mM) significantly stimulated the population of IPEC-J2 cells by 36% as determined by MTT assay. Adhesion level of LH511 was significantly increased by 9.2% when incubated with 5%LHFM_Cit-4mM and 5%LHFM_Cit-4mM reduced the adhesion of enterohemorrhagic (EHEC) and entero-invasive (EIEC) E. coli in IPEC-J2 cells by 35.79% and 42.74%, respectively. Treatment with 5%LHFM_Cit-4mM ameliorated lipopolysaccharide (LPS) from E. coli O55:B5 induced activated inflammatory cytokines expression (TNF-α, IL-6 and IL-8) and concentration (IL-6 and IL-8) and early apoptosis. It restored the transepithelial electrical resistance (TEER) and regulated the expression and distribution of tight junction (TJ) proteins (zonula occluden-1 (ZO-1), occludin and claudin-1), toll-like receptors (TLRs) (TLR2 and TLR4) and negative regulators of TLRs signalling pathway (A20 and IRAK-M). In conclusion, our findings suggested that 5%LHFM_Cit-4mM might have the positive effects on improving and maintaining the intestinal epithelial cell integrity and inflammatory response under both normal and pathogenic LPS-stimulated conditions.

Prevention of enteric bacterial infections and modulation of gut microbiota with conjugated linoleic acids producing Lactobacillus in mice.

Probiotics are recognized for outcompeting pathogenic bacteria by competitive receptor-mediated colonization and secretion of functional metabolites which are antimicrobial against certain microbes as well as improving host's gut health and immunity. Recently, we have constructed a bioactive Lactobacillus casei (LC) strain, LC+mcra , by inserting mcra (myosin cross-reactive antigen) gene, which stimulates the conversion of conjugated linoleic acids. In this study, we evaluated the modulation of gut microbiome and protective roles of LC+mcra against pathogenic Salmonella enterica serovar Typhimurium (ST) and enterohemorrhagic E. coli (EHEC) infections in BALB/cJ mice. We observed that LC+mcra colonized efficiently in mice gut intestine and competitively reduced the infection with ST and EHEC in various locations of small and large intestine, specifically cecum, jejunum, and ileum (p < 0.05). Positive modulation of the cecal microbiota, for example, higher relative abundances of Firmicutes, lower relative abundances of Proteobacteria, and increased bacterial species diversity/richness, was detected in ST-challenged mice pretreated with LC+mcra based on 16S metagenomic sequencing. Cytokine gene expression analysis indicated that mice pretreated with LC+mcra associated with attenuated bacterial pathogen-induced gut inflammation. Furthermore, mice fed daily with LC+mcra for one week could protect themselves from the impairments caused by enteric infections with ST or EHEC. These impairments include weight loss, negative hematological changes, intestinal histological alterations, and potential death. This in vivo study suggests that daily consumption of novel conjugated linoleic acids over-producing probiotic effectively improves intestinal microbiota composition and prevents/combats foodborne enteric bacterial infections with pathogenic Salmonella and diarrheagenic E. coli.

Improving bioreactor production of a recombinant glycoprotein in Escherichia coli: Effect of specific growth rate on protein glycosylation and specific productivity.

In the last decades bacterial glycoengineering emerged as a new field as the result of the ability to transfer the Campylobacter jejuni N- glycosylation machinery into Escherichia coli for the production of recombinant glycoproteins that can be used as antigens for diagnosis, vaccines, and therapeutics. However, the identification of critical parameters implicated in the production process and its optimization to jump to a productive scale is still required. In this study, we developed a dual expression glycosylation vector for the production of the recombinant glycoprotein AcrA-O157, a novel antigen that allows the serodiagnosis of the infection with enterohemorrhagic E. coli O157 in humans. Volumetric productivity was studied in different culture media and found that 2xYP had 6.9-fold higher productivity than the extensively used LB. Subsequently, bioreactor batch and exponential-fed-batch cultures were designed to determine the influence of the specific growth rate (µ) on AcrA-O157 glycosylation efficiency, production kinetics, and specific productivity. At µmax , AcrA glycosylation with O157-polysaccharide and the specific synthesis rate were maximal, constituting the optimal physiological condition for AcrA-O157 production. Our findings should be considered for the design, optimization, and scaling up of AcrA-O157 production and other recombinant glycoproteins attractive for industrial applications.

Long-Term Survival and Thermal Death Kinetics of Enterohemorrhagic Escherichia coli Serogroups O26, O103, O111, and O157 in Wheat Flour.

Wheat flour has been associated with outbreaks of enterohemorrhagic Escherichia coli (EHEC), but little is known on EHEC's survival during storage and thermal processing. The objective of this study was to determine long-term viability and thermal inactivation kinetics of EHEC serogroups O26, O103, O111, and O157. Wheat flour samples were inoculated with a cocktail of five strains of a single serogroup and stored at 23 and 35°C. Inoculated samples were heated at 55, 60, 65, and 70°C. Viability was determined by plate counting. Decimal reduction time (D) and first decimal reduction time (δ) values were calculated with log-linear and Weibull models, respectively. At 23°C, EHEC counts declined gradually for 84 days and samples tested positive from 84 to 280 days. The thermal resistance (D and δ) values ranged from 7.5 to 8.2 and 3.1 to 5.3 days, respectively, but there were no significant differences among serogroups (P ≤ 0.05). At 35°C, no EHEC was quantifiable by day 7 and no positive samples were detected after 49 days. Heating at 55 and 65°C resulted in δ-value ranges of 15.6 to 39.7 min and 3.0 to 3.9 min, respectively, with no significant difference among serogroups either. Z values were 12.6, 6.7, 10.2, and 13.4°C for O26, O103, O111, and O157, respectively. Thermal death kinetics of EHEC in flour were better described using the Weibull model. Survival and inactivation rates of four serogroups were remarkably similar. These findings indicated that all EHEC serovars tested remained viable for at least 9 months at room temperature and survived for up to 60 min at 70°C in wheat flour.IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) and Salmonella have recently caused several gastroenteritis outbreaks and recalls of wheat flour. Because EHEC can cause illness with very low doses and there is very scarce information regarding their ability to survive storage and heating in flour, the present study was undertaken to assess the long-term survival of EHEC serogroups O26, O103, O111, and O157 in flour. These findings are relevant, as we report that EHEC can survive for more than 9 months in wheat flour during storage. In addition, results obtained suggest that thermal inactivation at 65°C for 30 min or 2 months of storage at 35°C may be feasible strategies to mitigate the risk of most EHEC serovars in wheat flour.

The sialate O-acetylesterase EstA from gut Bacteroidetes species enables sialidase-mediated cross-species foraging of 9-O-acetylated sialoglycans.

The gut harbors many symbiotic, commensal, and pathogenic microbes that break down and metabolize host carbohydrates. Sialic acids are prominent outermost carbohydrates on host glycoproteins called mucins and protect underlying glycan chains from enzymatic degradation. Sialidases produced by some members of the colonic microbiota can promote the expansion of several potential pathogens (e.g. Clostridium difficile, Salmonella, and Escherichia coli) that do not produce sialidases. O-Acetyl ester modifications of sialic acids help resist the action of many sialidases and are present at high levels in the mammalian colon. However, some gut bacteria, in turn, produce sialylate-O-acetylesterases to remove them. Here, we investigated O-acetyl ester removal and sialic acid degradation by Bacteroidetes sialate-O-acetylesterases and sialidases, respectively, and subsequent utilization of host sialic acids by both commensal and pathogenic E. coli strains. In vitro foraging studies demonstrated that sialidase-dependent E. coli growth on mucin is enabled by Bacteroides EstA, a sialate O-acetylesterase acting on glycosidically linked sialylate-O-acetylesterase substrates, particularly at neutral pH. Biochemical studies suggested that spontaneous migration of O-acetyl esters on the sialic acid side chain, which can occur at colonic pH, may serve as a switch controlling EstA-assisted sialic acid liberation. Specifically, EstA did not act on O-acetyl esters in their initial 7-position. However, following migration to the 9-position, glycans with O-acetyl esters became susceptible to the sequential actions of bacterial esterases and sialidases. We conclude that EstA specifically unlocks the nutritive potential of 9-O-acetylated mucus sialic acids for foraging by bacteria that otherwise are prevented from accessing this carbon source.

Inhibitory potential of strepthonium A against Shiga toxin production in enterohemorrhagic Escherichia coli (EHEC) strain EDL933.

The production of shiga toxin (Stx) is a critical step in the establishment and progress of enterohemorrhagic Escherichia coli (EHEC) infections. The possible release of Stx from dead and dying bacteria, and the risk of resistance development have restricted the usage of antibiotics against EHEC. The chlorinated quaternary ammonium compound, strepthonium A, was isolated from the culture of Streptomyces sp. SBT345 that was cultivated from the Mediterranean sponge Agelas oroides. The structure was elucidated and confirmed by spectroscopic analyses including 1D and 2D NMR, ESI-HRMS, as well as ESI-HRMS2. Strepthonium A follows Lipinski's rule of five with respect to its molecular weight, CLogP values and the number of hydrogen acceptors and donors. Verotoxin ELISA assay demonstrated that Strepthonium A reduced the Stx production in EHEC strain EDL933 at 80 µM concentration without growth inhibition. This study demonstrates the potential of strepthonium A in restraining the production of Stx in EHEC infections.

Identification of a New Virulent Clade in Enterohemorrhagic Escherichia coli O26:H11/H- Sequence Type 29.

Enterohemorrhagic Escherichia coli (EHEC) O26 infections cause severe human diseases such as hemolytic uremic syndrome and encephalopathy, and is the predominant serogroup among non-O157 EHEC in many countries. Shiga toxin (Stx), which consists of two distinct types (Stx1 and Stx2), plays a central role in EHEC pathogenesis. The major stx gene type in EHEC O26 strains is stx1, although isolates with only stx2 have emerged in Japan since 2012 and have been reported in Europe. In this study, we selected 27 EHEC O26 strains isolated in Japan and identified a distinct genetic clade within sequence type (ST) 29, designated ST29C1, that carried only stx2 and had the plasmid gene profile ehxA+/katP-/espP+/etpD-. We showed that ST29C1 strains produced higher Stx2a levels, and greater virulence in Vero cells and in germ-free mice than other lineages. We also showed that ST29C1 was a distinct phylogenetic clade by SNP analysis using whole genome sequences and clearly differed from the major European EHEC O26 virulent clone, which was designated ST29C2 in this study. The combination of toxin production analysis, virulence analysis in Vero cells and germ-free mice, and phylogenetic analysis identified a newly emerging virulent EHEC clade.

Catabolite and Oxygen Regulation of Enterohemorrhagic Escherichia coli Virulence.

The biogeography of the gut is diverse in its longitudinal axis, as well as within specific microenvironments. Differential oxygenation and nutrient composition drive the membership of microbial communities in these habitats. Moreover, enteric pathogens can orchestrate further modifications to gain a competitive advantage toward host colonization. These pathogens are versatile and adept when exploiting the human colon. They expertly navigate complex environmental cues and interkingdom signaling to colonize and infect their hosts. Here we demonstrate how enterohemorrhagic Escherichia coli (EHEC) uses three sugar-sensing transcription factors, Cra, KdpE, and FusR, to exquisitely regulate the expression of virulence factors associated with its type III secretion system (T3SS) when exposed to various oxygen concentrations. We also explored the effect of mucin-derived nonpreferred carbon sources on EHEC growth and expression of virulence genes. Taken together, the results show that EHEC represses the expression of its T3SS when oxygen is absent, mimicking the largely anaerobic lumen, and activates its T3SS when oxygen is available through Cra. In addition, when EHEC senses mucin-derived sugars heavily present in the O-linked and N-linked glycans of the large intestine, virulence gene expression is initiated. Sugars derived from pectin, a complex plant polysaccharide digested in the large intestine, also increased virulence gene expression. Not only does EHEC sense host- and microbiota-derived interkingdom signals, it also uses oxygen availability and mucin-derived sugars liberated by the microbiota to stimulate expression of the T3SS. This precision in gene regulation allows EHEC to be an efficient pathogen with an extremely low infectious dose. IMPORTANCE: Enteric pathogens have to be crafty when interpreting multiple environmental cues to successfully establish themselves within complex and diverse gut microenvironments. Differences in oxygen tension and nutrient composition determine the biogeography of the gut microbiota and provide unique niches that can be exploited by enteric pathogens. EHEC is an enteric pathogen that colonizes the colon and causes outbreaks of bloody diarrhea and hemolytic-uremic syndrome worldwide. It has a very low infectious dose, which requires it to be an extremely effective pathogen. Hence, here we show that EHEC senses multiple sugar sources and oxygen levels to optimally control the expression of its virulence repertoire. This exquisite regulatory control equips EHEC to sense different intestinal compartments to colonize the host.

A secretome view of colonisation factors in Shiga toxin-encoding Escherichia coli (STEC): from enterohaemorrhagic E. coli (EHEC) to related enteropathotypes.

Shiga toxin-encoding Escherichia coli (STEC) regroup strains that carry genes encoding Shiga toxin (Stx). Among intestinal pathogenic E. coli, enterohaemorrhagic E. coli (EHEC) constitute the major subgroup of virulent STEC. EHEC cause serious human disease such as haemorrhagic colitis and haemolytic-uremic syndrome. While EHEC have evolved from enteropathogenic E. coli, hybrids with enteroaggregative E. coli have recently emerged. Of note, some enteroinvasive E. coli also belong to the STEC group. While the LEE (locus of enterocyte effacement) is a key and prominent molecular determinant in the pathogenicity, neither all EHEC nor STEC contain the LEE, suggesting that they possess additional virulence and colonisation factors. Currently, nine protein secretion systems have been described in diderm-lipopolysaccharide bacteria (archetypal Gram-negative) and can be involved in the secretion of extracellular effectors, cell-surface proteins or assembly of cell-surface organelles, such as flagella or pili. In this review, we focus on the secretome of STEC and related enteropathotypes, which are relevant to the colonisation of biotic and abiotic surfaces. Considering the wealth of potential protein trafficking mechanisms, the different combinations of colonisation factors and modulation of their expression is further emphasised with regard to the ecophysiology of STEC.

Increased EHEC survival and virulence gene expression indicate an enhanced pathogenicity upon simulated pediatric gastrointestinal conditions.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) are major foodborne pathogens that constitute a serious public health threat, mainly in young children. Shiga toxins (Stx) are the main virulence determinants of EHEC pathogenesis but adhesins like intimin (eae) and Long polar fimbriae (Lpf) also contribute to infection. The TNO GastroIntestinal Model (TIM) was used for a comparative study of EHEC O157:H7 survival and virulence under adult and child digestive conditions. METHODS: Survival kinetics in the in vitro digestive tract were determined by plating while bacterial viability was assessed by flow cytometry analysis. Expression of stx, eae, and lpf genes was followed by reverse transcriptase-quantitative PCR (RT-qPCR) and Stx production was measured by ELISA (enzyme-linked immunosorbent assay). RESULTS: Upon gastrointestinal passage, a higher amount of viable cells was found in the simulated ileal effluents of children compared to that of adults (with 34 and 6% of viable cells, respectively). Expression levels of virulence genes were up to 125-fold higher in children. Stx was detected only in child ileal effluents. CONCLUSION: Differences in digestive physicochemical parameters may partially explain why children are more susceptible to EHEC infection than adults. Such data are essential for a full understanding of EHEC pathogenesis and would help in designing novel therapeutic approaches.

Global transcriptional regulation by H-NS and its biological influence on the virulence of Enterohemorrhagic Escherichia coli.

As a global transcriptional regulator, H-NS, the histone-like nucleoid-associated DNA-binding and bridging protein, plays a wide range of biological roles in bacteria. In order to determine the role of H-NS in regulating gene transcription and further find out the biological significance of this protein in Enterohemorrhagic Escherichia coli (EHEC), we conducted transcriptome analysis of hns mutant by RNA sequencing. A total of 983 genes were identified to be regulated by H-NS in EHEC. 213 and 770 genes were down-regulated and up-regulated in the deletion mutant of hns, respectively. Interestingly, 34 of 97 genes on virulence plasmid pO157 were down-regulated by H-NS. Although the deletion mutant of hns showed a decreased survival rate in macrophage compared with the wild type strain, it exhibited the higher ability to colonize mice gut and became more virulent to BALB/c mice. The BALB/c mice infected with the deletion mutant of hns showed a lower survival rate, and a higher bacterial burden in the gut, compared with those infected with wild type strain, especially when the gut microbiota was not disturbed by antibiotic administration. These findings suggest that H-NS plays an important role in virulence of EHEC by interacting with host gut microbiota.

Effect of Direct-Fed Microbial Dosage on the Fecal Concentrations of Enterohemorrhagic Escherichia coli in Feedlot Cattle.

Contamination of beef products by Shiga toxin-producing Escherichia coli is a concern for food safety with a particular subset, the enterohemorrhagic E. coli (EHEC), being the most relevant to human disease. To mitigate food safety risks, preharvest intervention strategies have been implemented with the aim to reduce EHEC in cattle. One class of interventions that has been widely used in feedlots is direct-fed microbials (DFMs), which can contain various dosing rates of probiotic bacteria. Here we compare the use of two different doses of a commercially available DFM on total EHEC load in a commercial feedlot setting. The DFMs used were the standard 10(9) Propionibacterium freudenreichii and 10(6) Lactobacillus acidophilus colony forming units (CFUs)/head/day dose of Bovamine(®) (Nutrition Physiology Company, Guymon, OK) and the higher dose, Bovamine Defend™ (Nutrition Physiology Company), which is dosed at 10(9) P. freudenreichii and 10(9) Lactobacillus acidophilus CFUs/head/day. To analyze the total EHEC fecal concentration, 2200 head of cattle were assigned a DFM feed regimen lasting approximately 5 months. At harvest, 480 head of cattle were sampled using rectoanal mucosal swabs. A quantitative polymerase chain reaction assay targeting ecf1 was used to enumerate the total EHEC fecal concentration for 240 head fed the low-dose DFM and 240 head fed the high-dose DFM. No significant difference (p > 0.05) in the fecal concentration of total EHEC was observed between the two doses. This suggests that using an increased dosage provides no additional reduction in the total EHEC fecal concentration of feedlot cattle compared to the standard dosage.

Enterohemorrhagic Escherichia coli infection has donor-dependent effect on human gut microbiota and may be antagonized by probiotic yeast during interaction with Peyer's patches.

Enterohemorrhagic Escherichia coli (EHEC) are major food-borne pathogens responsible for serious infections ranging from mild diarrhea to hemorrhagic colitis and life-threatening complications. Shiga toxins (Stxs) are the main virulence factor of EHEC. The antagonistic effect of a prophylactic treatment with the probiotic strain Saccharomyces cerevisiae against EHEC O157:H7 was investigated using complementary in vitro human colonic model and in vivo murine ileal loop assays. In vitro, the probiotic treatment had no effect on O157:H7 survival but favorably influenced gut microbiota activity through modulation of short-chain fatty acid production, increasing acetate production and decreasing that of butyrate. Both pathogen and probiotic strains had individual-dependent effects on human gut microbiota. For the first time, stx expression was followed in human colonic environment: at 9 and 12 h post EHEC infection, probiotic treatment significantly decreased stx mRNA levels. Besides, in murine ileal loops, the probiotic yeast specifically exerted a trophic effect on intestinal mucosa and inhibited O157:H7 interactions with Peyer's patches and subsequent hemorrhagic lesions. Taken together, the results suggest that S. cerevisiae may be useful in the fight against EHEC infection and that host associated factors such as microbiota could influence clinical evolution of EHEC infection and the effectiveness of probiotics.

Development of a quantitative real time PCR assay to detect and enumerate Escherichia coli O157 and O26 serogroups in bovine recto-anal swabs.

Escherichia coli O157 and O26 shedding patterns in cattle are known to vary widely. To address gaps in the understanding of the underlying factors which impact on shedding dynamics, sensitive and rapid quantitative methods which can be applied in surveillance studies on cattle are required. Current approaches for enumeration of verocytotoxigenic E. coli (VTEC) in cattle faeces are based on direct plating onto selective agars, most probable number (MPN) or real time PCR applied directly to faecal samples, all of which have limitations in terms of the labour involved or their sensitivity. The objective of this study was to develop a sensitive real time quantitative PCR assay, to quantify O157 and O26 in bovine recto-anal junction (RAJ) swabs. The approach was to target serogroup specific genes rfbE and wzx, and to couple a short enrichment, with the use of a standard calibration curve relating real time PCR cycle threshold (Ct) values against the initial concentration of the pathogen in the sample. Following initial experiments in broth culture, a 5h enrichment in modified tryptone soya broth with novobiocin (20 mg/l) (mTSBn) was found to be optimal, and a linear correlation between inocula (Log10 1 to 6 CFU ml(-1)) and the PCR Ct values for both E. coli O157 (R(2)=0.99, rsd=0.58) and E. coli O26 (R(2)=0.99, rsd=0.44) was confirmed. The developed method was then applied to bovine RAJ swab samples (n=153), which were inoculated with E. coli O157 or O26 (Log10 1 to 7 CFU swab(-1)). Calibration curves yielded correlations for E. coli O157 of R(2)=0.86, rsd=0.72 and for O26 (R(2)=0.88, rsd=0.69). In conclusion, a sensitive method for detection and enumeration of two significant VTEC serogroups in bovine RAJ samples has been developed and validated, and will support studies on the bovine shedding dynamics of these pathogens in cattle.

Survival of pathogenic enterohemorrhagic Escherichia coli (EHEC) and control with calcium oxide in frozen meat products.

This study investigated both the level of microbial contamination and the presence of enterohemorrhagic Escherichia coli (EHEC) in frozen meat products, followed by the evaluation of its survival over 180 days under frozen temperature. We also examined the effect of calcium oxide on the populations of EHEC, E. coli O157:H7 and EPEC under both 10 °C and -18 °C storage conditions. Afterward, the morphological changes occurring in EHEC cells in response to freezer storage temperature and calcium oxide (CaO) treatments were examined using transmission electron microscopy. Among the frozen meat products tested, the highest contamination levels of total aerobic counts, coliforms and E. coli were observed in pork cutlets. Examination showed that 20% of the frozen meat products contained virulence genes, including verotoxin (VT) 1 and 2. Over 180 days of frozen storage and after 3 freeze-thaw cycles, the population of EHEC did not change regardless of the type of products or initial inoculated concentration, indicating the strong survival ability of EHEC. Subsequent testing revealed that the growth of three pathogenic E. coli strains was completely inhibited in meat patties prepared with 1% CaO, stored at 10 °C. However, the addition of 2% CaO was necessary to control the survival of EHEC, E. coli O157:H7 and EPEC in meat patties stored at -18 °C. CaO reduced the population of E. coli O157:H7 more effectively than the other EHEC and EPEC strains at both 10 °C and -18 °C. Transmission electron microscopy analysis revealed that exposed EHEC cells were resistant to the freezer storage temperature, although some cells incurred injury and death after several freeze-thaw cycles. Most of the cells exposed to CaO were found to have died or lost their cellular integrity and membranes, indicating that CaO has the potential to be used as a powerful antimicrobial agent for manufacturing frozen meat products.

Colonization of the meat extracellular matrix proteins by O157 and non-O157 enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) are anthropozoonotic agents that range third among food-borne pathogens respective to their incidence and dangerousness in the European Union. EHEC are Shiga-toxin producing E. coli (STEC) responsible for foodborne poisoning mainly incriminated to the consumption of contaminated beef meat. Among the hundreds of STEC serotypes identified, EHEC mainly belong to O157:H7 but non-O157 can represent 20 to 70% of EHEC infections per year. Seven of those serogroups are especially of high-risk for human health, i.e. O26, O45, O103, O111, O121, O145 and O104. While meat can be contaminated all along the food processing chain, EHEC contamination essentially occurs at the dehiding stage of slaughtering. Investigating bacterial colonization to the skeletal-muscle extracellular matrix (ECM) proteins, it appeared that environmental factors influenced specific and non-specific bacterial adhesion of O157 and non-O157 EHEC as well as biofilm formation. Importantly, mechanical treatment (i.e. shaking, centrifugation, pipetting and vortexing) inhibited and biased the results of bacterial adhesion assay. Besides stressing the importance of the protocol to investigate bacterial adhesion to ECM proteins, this study demonstrated that the colonization abilities to ECM proteins vary among EHEC serogroups and should ultimately be taken into consideration to evaluate the risk of contamination for different types of food matrices.